Dormancy and Persistence:
Bacteria require specific conditions for growth. Under ideal conditions they grow exponentially but under limiting conditions they stop dividing. In such conditions bacteria are known to pass into a state where the metabolic activity is minimized. In some bacteria this state is achieved through sporulation but in others a functionally similar state known as dormancy takes over. Dormant bacteria do not give rise to colonies but if incubated under appropriate conditions it is possible to resuscitate them. An important attribute of dormant bacteria is that they are more equipped to persist under adverse conditions, such as during drug therapy, than actively growing bacteria. Mycobacterium tuberculosis, the tuberculosis (TB) pathogen, is particularly known to persist in a dormant state for a long time within human hosts. In the context of mycobacterial infections the terms “dormant” and “persistent” are generally used synonymously to describe the non-replicating bacteria. In tuberculosis the term latency is often used. It means that following primary infection an active disease may not develop. The infection may remain latent for a prolonged period of time until at some stage reactivation occurs for reasons that are not clearly known as yet. However there are evidences to show that the latency is closely related to dormancy and persistence.
Chaperon Function:
Proteins are known to have specific functions which arise out of their secondary and tertiary structures. Polypeptides may attain these structures by themselves, but in most cases the process of attaining such structures termed as “folding” is aided by other proteins which are functionally known as “chaperons”. The chaperons may not only aid folding but also they may prevent misfolding. The ability to prevent misfolding is important as such misfolded proteins form nonviable aggregates which are lethal to the cell. The ability of chaperons to prevent aggregation can be reproduced in vitro and this is often used as a measure of chaperon activity. In most cases the synthesis of the chaperons is increased several fold by heat shock. The increased synthesis during heat shock is essential as under such conditions proteins are more likely to be denatured. Chaperons are therefore often referred to in a loose sense as “heat shock proteins” or Hsps. Hsps need not always be induced by heat shock. Other shocks such as acid shock, low oxygen tension etc. can also bring about the same effect.
Peptide Inhibitors:
Peptides are polymers of amino acids which are linked through amide bonds. The smallest peptide that can be formed is a di-peptide. Peptides may however be larger comprising of as many as forty amino acids. Peptides can exist in specific conformations and can have biological activity. They may act as hormones, immuno-modulators, antibiotics, antigens, agonists as well as antagonists of various functions. The ability to synthesize peptides chemically and incorporate within them uncommon amino acids make them useful systems for obtaining novel bio-active molecules. Because of their conformational flexibility peptides can mimic the structures of natural ligands. Such peptide mimics can be used as inhibitors of biological processes.
Lead Compounds:
Present day drug development processes employ either random high-throughput techniques or structure based drug design methods. In either case the initial screens lead to compounds that cannot be used directly as drugs but they can be potentially developed further after studying the manner in which they interact with the target. In other words an initial low affinity interaction can be converted to a high affinity interaction. The compounds obtained after initial screening thus serve as “lead compounds” that can be developed further into potential drugs.
Pathogenic mycobacteria are the causative agents of a number of human and animal diseases. For example, tuberculosis is a health problem of considerable importance in the human population. Recent estimates are that as much as one-third of the population of the world is infected with M. tuberculosis, that there are 30 million active cases, that there are some 10 million new cases annually and that TB causes some 6 percent of all deaths worldwide (see e.g., Dye et al., 1999). Despite availability of chemotherapeutic agents, persistence and multi-drug resistance make it difficult to eliminate M. tuberculosis as a major health threat using currently available intervention strategies. It is known that M. tuberculosis survives within the hostile environment of macrophages and it is difficult to eliminate this form completely. Identification of new classes of drugs, which are active against latent TB, is thus considered imperative.
Development of novel drugs against TB has become a challenging area of research because of the unusual ability of the TB pathogen (M. tuberculosis) to resist drugs. Such resistance arises not only due to mutations but also due to the ability of the pathogen to enter into a dormant phase in which it can persist for prolonged periods of time (see e.g., Stewart et al., 2003). This happens particularly when it is encapsulated within a granuloma—a structure formed by the activated monocyte—macrophage system of the host. The conditions within the granuloma are far from ideal for mycobacterial growth. In particular, M. tuberculosis is an aerobic organism, whereas the conditions within the granuloma are highly anaerobic. Although under such conditions active growth is halted, the bacteria can persist indefinitely by entering into a dormant phase. Drug therapy further accelerates the shift from the active to the dormant or persistent phase (Coates et al., 2002). Treatment with the presently available drugs therefore can potentially cause the accumulation of dormant bacilli, which can reactivate themselves at a later stage. The dormant bacilli are therefore the major cause of concern as it leads to persistence of TB, which cannot be cured easily.
The persistent state can be mimicked under laboratory conditions by growing M. tuberculosis to stationary phase or growing the organism under hypoxic conditions or nutrient deprivation (Yuan et al., 1996; Sherman et al., 2001; Betts et al., 2002). It has been found that under these conditions the expression of a large number of genes are induced which are possibly required for the viability of the organism in the persistent phase. The proteins induced in the persistent phase can be considered as drug targets for preventing persistent TB. The 16 kDa alpha-crystallin like heat-shock protein, Hsp16.3 is an extremely important component of the dormant phase metabolism of the pathogen. It has also been demonstrated that over-expression of this protein in wild-type M. tuberculosis resulted in a slower decline in viability following the end of log-phase (Yuan et al., 1996). The protein has been demonstrated to be able to inhibit the thermal denaturation of various other proteins (Chang et al., 1996). From these evidences it has been suggested that the Hsp16.3 may play a role in enhancing long-term protein stability and therefore long-term survival in mycobacteria.
More recently the regulation of the M. tuberculosis hypoxic response has been elucidated. Under hypoxic conditions the expression of an operon encoding two polypeptides, which are constituents of a two-component signaling system (Rv3133c/3132c), is induced. Gene disruption experiments indicate that the induction of Hsp16.3 gene expression under hypoxic conditions is severely impaired (Sherman et al., 2001). This indicates that Hsp16.3 is a key player in the hypoxic response.
Hsp16.3 belongs to a family of proteins known as alpha-crystallin (Acr), which play an important role in the maintenance of the transparency of vertebrate eye. The primary function of these proteins is to act as molecular chaperons. Most of bacterial homologues of this protein have been found to play an important role in spore formation in Bacillus subtilis and are induced in response to acute stresses in other microorganisms. Initially the M. tuberculosis Acr (Hsp16.3) protein was characterized as a major membrane protein but subsequent work has revealed that it is a potent ATP-independent chaperon whose complex oligomeric active structure consists of trimer of trimers (Chang et al., 1996).
Hsp16.3 expression has also been found to be induced during the course of in vitro infection of macrophages. When the gene for Hsp16.3 (acr) of M. tuberculosis was replaced with a hygromycin cassette by allelic exchange in M. tuberculosis H37Rv, the resulting strain was shown to be equivalent to wild type H37Rv in in vitro growth rate and infectivity but was significantly impaired for growth in both mouse bone marrow derived macrophages and THP-1 cells (Yuan et al., 1998). These results indicate that Hsp16.3 plays an important role in maintenance of long-term viability during latent, asymptotic infections. That Hsp 16.3 is at least one of the key virulence factors has been clearly demonstrated by eliminating or down regulating the expression of the alpha-crystallin heat shock protein gene (acr) (Barry III et al., U.S. Pat. No. 6,403,100). As a result the virulent strain of M. tuberculosis became attenuated, which means that it lost its virulence substantially, indicating that Hsp16.3 is one of the key players of mycobacterial virulence. Given this background it is clear that the effective inhibitors against the Hsp16.3 protein would be of significant importance to combat persistent TB.
Peptides are considered as useful therapeutic agents as they can specifically interfere with protein-protein interaction. Nature abounds in various antimicrobial peptides (see e.g., the data base ANTIMIC: http://research.i2r.a-star.edu.sq/Templar/DB/ANTIMIC/). Some of these such as defensins act by interacting at the level of membrane permeabilization. However there are others like pyrrhocoricin (Otvos et al., 2000), which have specific cellular targets such as Hsp70 or DnaK, which like Hsp16.3, is a chaperon. Pyrrhocoricin specifically inhibits DnaK from bacterial sources such as E. coli but not human and hence examples such as this indicate that although chaperons are conserved in evolution there exists peptide binding pockets specifically present in bacterial chaperons, which can be targeted for drug design. Since naturally occurring peptide inhibitors of Hsp16.3 are as yet not known hence an alternative strategy of identifying peptide inhibitors through selection from a pool of phage displayed peptides has been developed as claimed in this disclosure.